The human pathogen Chlamydia trachomatis is a significant concern in the United States due to its prevalence and the combined health and socioeconomic impact of acute and chronic disease. Chlamydiae are obligate intracellular pathogens and possess the ability to modulate host-cell functions while sequestered within a membrane-bound vacuole. Expression of a virulence-associated type III secretion system (T3SS) represents one mechanism employed to modulate critical host cell pathways. During the past funding cycle, we identified multiple chlamydial T3S substrates capable of influencing these host cellular processes. The C. trachomatis locus containing the identified effector protein CT694 contains multiple substrates that are deployed by infectious particles during or subsequent to the invasion process. We propose to elucidate molecular mechanisms regarding the anti-host activities of these proteins and delineate the consequences of effector activity on the ability of Chlamydiae to establish and maintain a specialized intracellular niche. A combination of methods designed to identify interactions of chlamydial proteins with host targets will be employed to establish relevant functions. The consequences of these interactions will be investigated in both a tissue culture infection model and a murine model of acute chlamydial infection. We furthermore propose to evaluate whether differences in these effectors among chlamydial species account for any of the distinct, species-specific events related to early chlamydial development. The chlamydial type III secretion system represents an attractive, yet relatively unexplored, mechanism to achieve modulation of host cell activities. Given the comparative difficulty associated with study of obligate intracellular bacteria, investigation of host pathways specifically targeted by the type III secretion mechanism continues to represent a productive approach to elucidate novel pathogenic mechanisms. These studies will lead to an enhanced understanding of Chlamydia-mediated disease and have the potential to yield novel preventative and treatment therapies.